Moreover, forced attachment of dendrites to the ECM by integrin overexpression in these tiling mutants rescued both isoneuronal and heteroneuronal dendritic crossing defects. Our results demonstrate the importance of

2D distribution in the tiling of see more class IV da neurons and reveal that the TORC2/Trc pathway plays a major role in ensuring tiling by confining dendrites to a 2D space rather than by mediating homotypic repulsion. The Drosophila larval body wall is evenly covered by class IV da dendrites ( Figure 1A). To understand how dendrites and epidermal cells are arranged relative to one another in a 3D space, we simultaneously imaged class IV da dendrites and the ECM of the epidermis in live third instar larvae by labeling dendrites with an improved class IV-specific membrane marker ppk-CD4-tdTom ( Han et al., 2011; see Experimental Procedures) and the ECM with viking-GFP (vkg-GFP). Since the epidermis is comprised of a thin layer of epithelial cells, a detailed cross-section view of the epidermis requires 3D reconstruction of an image stack with a high resolution on the z axis (i.e., the apical-basal axis of epidermal cells, Figure 1B). To maximize the resolving power on the z axis, we adopted a high-resolution confocal imaging protocol combined with deconvolution

(see Experimental Procedures), which greatly improves the visualization of the spatial relationship BMS-387032 in vivo between dendrites and the ECM. Each abdominal hemisegment of the Drosophila larva contains three class IV da neurons, the dorsal ddaC, the ventro-lateral v′ada, and the ventral vdaB ( Figure 1A). We examined the dendrite positioning of all three neurons along the z axis. Most of the dendrites were found to directly contact the ECM (green dendrites in Figures 1C′, 1D′, and 1E′; also see Movie S1 available online), while a small percentage of dendrites are detached from the ECM Isotretinoin in the apical direction (magenta dendrites in

Figures 1C′, 1D′, and 1E′). Since epidermal cells are located immediately apical to the ECM ( Figure 1B), the dendrites detached from the ECM are enclosed within the epidermal layer (referred to as enclosed dendrites hereafter). We noticed several characteristics of these enclosed dendrites. First, they can be either segments in the middle of stabilized branches (small panel 1 in Figures 1C′, 1D′, and 1E′), or parts of terminal dendrites (small panel 2 in Figures 1C′, 1D′, and 1E′). Second, the enclosed dendrites may appear to cross other branches attached to the ECM in z axis projections, even though the overlapping dendrites are located at different depths in the epidermis and are not in direct contact (small panel 3 in Figures 1C′, 1D′, 1E′, and Figures 1F–1F″). We call this type of dendritic overlap noncontacting crossing.